RESEARCH SUMMARY The goal of this project is to understand the role of PTEN in DNA metabolism and replication. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is one of the most frequently lost or mutated tumor suppressors in cancer, commonly lost in endometrial cancer, glioblastoma, breast cancer, and prostate cancer. PTEN is a dual-specificity phosphatase whose main substrate is the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), and PTEN activity negatively regulates the phosphoinositide 3- kinase (PI3K)/AKT growth signaling pathway. Work in our lab and others has shown that the deletion of both alleles of PTEN causes increased growth rate and proliferation in a variety of normal tissues, including in primary mouse embryonic fibroblasts (MEFs). Additionally, we have demonstrated a glutamine dependency, an increased glutamine flux into de novo pyrimidine synthesis, and an increased sensitivity to pharmacologic inhibition of de novo pyrimidine synthesis in primary Pten?/? MEFs. We propose that these results implicate the nucleotide metabolism, the cell cycle, and DNA replication as potential avenues through which the loss of PTEN could contribute to deregulated growth. We posit to explore these pathways using primary Ptenflox/flox MEFs as a model system. In Aim 1, we will investigate how the loss of PTEN expression sensitizes cells to inhibition of de novo pyrimidine synthesis through altered cell cycle dynamics. To investigate the molecular basis of this sensitivity, we will examine DNA damage and replication stress, cell death, and cell cycle checkpoint activation after pharmacologic inhibition of de novo pyrimidine synthesis in primary Pten?/? MEFs. Given the potential use of leflunomide for patients with PTEN-deficient tumors, a comprehensive understanding of the cellular consequences of leflunomide treatment in the context of PTEN loss will enable more effective use. In Aim 2, we will characterize the impact of PTEN loss on the duration of S phase and DNA replication efficiency. To determine the length of S phase in primary Pten?/? MEFs, we will use two experimental methods that allow for the determination of S phase length: dual-pulse DNA replication labeling with thymidine analogs and live cell imaging in combination with fluorescent-labeled cell cycle-specific proteins. To investigate DNA replication efficiency, we will perform fiber combing to directly visualize replicating DNA. Knowledge of the mechanism behind altered replication dynamics in the context of PTEN loss would contribute to the basic understanding of the mechanisms of evading growth regulation in cancer cells.